Controlled release of growth factors and signaling molecules for promoting angiogenesis

ABSTRACT

The present invention comprises compositions, methods, and devices for delivering angiogenic factors and signaling molecules to a target tissue, and controlling the release of these factors and signaling molecules to spatially and temporally restrict their release and dissemination, for the purpose of promoting angiogenesis in target tissues.

RELATED APPLICATIONS

This application is a national stage application, filed under 35 U.S.C.§371, of International Application No. PCT/US2009/045856, filed Jun. 1,2009, which claims the benefit of provisional application U.S. Ser. No.61/130,486, filed May 30, 2008, the contents of which are incorporatedby reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with Government support under RO1 HL069957awarded by the National Institutes of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to the fields of cardiology, tissuerepair, and preventative medicine.

BACKGROUND OF THE INVENTION

Angiogenesis refers to a process of new blood vessel formation. Subjectssuffering from coronary arterial disease (CAD) and peripheral arterialdisease (PAD) can be treated by promoting angiogenesis in the tissuelacking sufficient blood flow. However, current methods of administeringangiogenic drugs are sub-optimal because they cannot control thepresentation of multiple compounds separately. Moreover, systemicadministration of drugs at concentrations that are therapeuticallyeffective for the affected area cause surrounding, healthy, tissues tobe exposed unnecessarily to pro-angiogenic growth factors and could leadto undesirable side effects.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for controllingthe local presentations of pro-angiogenic growth factors and signalingmolecules that are together used to achieve angiogenesis at the tissueor organ of interest. The compositions and methods of the presentinvention allow the concerted and joint presentations of deliveredgrowth factors and signaling molecules to be controlled separately bychanging physical and/or chemical properties of the polymer deliverymaterial to achieve an appropriate local concentration of thefactor/molecules at the target tissue site.

Subjects to be treated in the manner described herein have beendiagnosed as suffering from or at risk of developing an ischemiccondition. The methods are suitable to treatment of human patients, aswell as being suitable for veterinary use (e.g., treatment of companionanimal such as dogs and cats. In a preferred embodiment, the methods areused to develop treatments for chronic ischemia in coronary andperipheral artery disease for diabetic subjects. Alternatively, or inaddition, the present invention is used to improve wound healing inulcers for diabetic subjects. The impaired endothelial tissues ofdiabetic subjects often have a reduced response to regularpro-angiogenic factors, making the continuous activation of vasculargrowth induced by the sustained and distinct presentation of growthfactors and signaling molecules provided by the present inventionparticularly valuable. This type of continuous, sustained, and distinctpresentation of pro-angiogenic factors and signaling molecules is notcurrently possible. Current methods that attempt to achieve similartherapeutic outcomes also induce deleterious side-effects due tosubstantial systemic dissemination of these factors throughout the body.

A device, which overcomes the shortcomings of existing approaches,comprises a scaffold composition, a bioactive composition, and aregulatory agent. The bioactive composition and the regulatory agent areincorporated into or coated onto the scaffold, e.g., a polymeric gel,composition, and the regulatory agent controls the activity of thebioactive agent. The bioactive composition is released from the scaffoldcomposition at a first rate and the regulatory agent is released fromsaid scaffold composition at a second rate. For example, the bioactivecomposition exits from the scaffold composition slower or fasterrelative to the regulatory agent. For example, vascular endothelialgrowth factor (VEGF) as a bioactive composition exits from the scaffoldcomposition for a time period of one or more, e.g., 4 weeks, while gammasecretase inhibitor as a regulatory agent exits from the same scaffoldcomposition for a time period of one or more days, e.g., 3 days. Thedifference between the release rates of multiple factors is important indetermining the final angiogenesis outcomes. For example, the desiredtime-frame for Notch inhibitor delivery will be 7-20 fold (e.g., 8, 10,15, or 18-fold) shorter than that of VEGF or PDGF, i.e., Notch inhibitoris released within 1-3 days while VEGF or PDGF is released within 7-60days (e.g., 7, 10, 15, 30, 45, or 60 days). An exemplary bioactivecomposition is a pro-angiogenic factor such as VEGF and/or PDGF and anexemplary regulatory agent is an inhibitor or enhancer of angiogenesissuch as DAPT.

One or more bioactive compositions are incorporated into or coated ontothe scaffold composition, and the scaffold composition temporallycontrols release of the bioactive composition. Alternatively, or inaddition, the scaffold composition spatially controls release of abioactive composition. In another embodiment, the bioactive compositionincorporated into or coated onto the scaffold composition temporally orspatially regulates release of a second bioactive composition.

The present invention also comprises a method of inducing blood vesselgrowth in a target tissue of a mammal, comprising providing a devicecomprising a scaffold composition with a bioactive composition beingincorporated therein or thereon and contacting a mammalian tissue withthe device wherein said scaffold composition temporally controls releaseof the bioactive composition and wherein the bioactive compositioninduces angiogenesis within the target tissue. For example, a polymericgel composition loaded with pro-angiogenic factors and a regulatoryfactor is injected directly into the target site, or into a site that isadjacent to or in close proximity to the target site in whichangiogenesis is desired. For example, the site of administration is 10mm, 25 mm, 50 mm, 1 cm, 5 cm, 10 cm, 50 cm from the target tissue sitewhere angiogenesis is to occur. In addition, multiple simultaneousinjections (in different spatial locations, e.g., encircling orsurrounding an affected anatomical location/site), or repeatinginjections every 1-2 weeks, at the same site or every few mm or cm apartin an ischemic region may be desirable.

The present invention further comprises a method of augmenting bloodvessel growth, comprising providing a device comprising a scaffoldcomposition with a bioactive composition being incorporated therein orthereon and contacting a mammalian tissue with the device, wherein saidscaffold composition temporally controls release of the bioactivecomposition and wherein the bioactive composition induces growth fromexisting blood vessels.

The bioactive composition of the devices of the present invention isnon-covalently linked to said scaffold composition. Alternatively, thebioactive composition is covalently linked to said scaffold composition.

Bioactive compositions of the present invention consist of, consistessentially of, or comprise one or more factors, which are administeredeither by direct protein delivery or delivering gene sequences to havecells locally make proteins. Exemplary bioactive compositions caninclude receptor ligands, transcription factors, and/or regulatorymolecules.

Receptor ligands include, but are not limited to, vascular endothelialgrowth factor (VEGF (A-F)), fibroblast growth factors (acidic and basicFGF 1-10), granulocyte-macrophage colony-stimulating factor (GM-CSF),insulin, insulin growth factor or insulin-like growth factor (IGF),insulin growth factor binding protein (IGFBP), placenta growth factor(PIGF), angiopoietin (Ang1 and Ang2), platelet-derived growth factor(PDGF), hepatocyte growth factor (HGF), transforming growth factor(TGF-α, TGF-β, isoforms 1-3), platelet-endothelial cell adhesionmolecule-1 (PECAM-1), vascular endothelial cadherin (VE-cadherin),nitric oxide (NO), chemokine (C-X-C motif) ligand 10 (CXCL10) or IP-10,interleukin-8 (IL-8), hypoxia inducible factor (HIF), monocytechemotactic protein-1 (MCP-1), vascular cell adhesion molecule (VCAM),ephrin ligands (including Ephrin-B2 and -B4).

Transcription factors include, but are not limited to, HIF-1α, HIF-1βand HIF-2α, Ets-1, Hex, Vezf1, Hox, GATA, LKLF, COUP-TFII, Hox, MEF2,Braf, Prx-1, Prx-2, CRP2/SmLIM and GATA family members, basichelix-loop-helix factors and their inhibitors of differentiation.

Regulatory molecules include, but are not limited to, enzymes (matrixmetalloproteinase (MMP), tissue plasminogen activator (PLAT or tPA),cyclooxygenase (COX), angiogenin), molecules regulating Notch signalingwhich consists of, consists essentially of, or comprises monoclonalantibodies to Notch ligands and receptors, RNA interference, antisenseNotch, receptor and mastermind-like 1 (MAML1) decoys, beta andgamma-secretase inhibitors (GSI), or any other molecules that canactivate or inhibit Notch signaling.

Devices of the present invention consist of, consist essentially of, orcomprise one or more bioactive compositions. A second bioactivecomposition consists of, consists essentially of, or comprises asignaling molecule selected from the group consisting of monoclonalantibodies to Notch ligands and receptors, RNA interference, antisenseNotch, receptor and mastermind-like 1 (MAML1) decoys, beta andgamma-secretase inhibitors (GSI), or any other molecules that canactivate or inhibit Notch signaling.

Alternatively, or in addition, signaling molecules of the secondbioactive composition are selected from the group of any other moleculesthat can inhibit or activate Notch signaling. In a preferred embodimentof the present invention, the signaling molecule is DAPT(N—[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester)(Sigma-Aldrich, St. Louis, Mo.). Signaling molecules of the secondbioactive composition are released from scaffolds and devicessimultaneously or sequentially with each other. Signaling molecules ofthe second bioactive composition are released from scaffolds and devicessimultaneously or sequentially with bioactive compositions comprisingangiogenic factors. Preferably, the regulatory molecule is a Notchinhibitor such as the gamma secretase inhibitor DAPT.

Scaffold compositions of the present invention degrade at apredetermined rate based on a physical parameter selected from the groupconsisting of temperature, pH, hydration status, and porosity.Alternatively, or in addition, scaffold compositions are enzymaticallydigested by a composition elicited by a contacting cell, said release ofsaid bioactive composition being dependent upon the rate of enzymaticdigestion. A contacting cell is defined as a cell belonging to a targetcell wherein the scaffold composition or device resides that physicallycontacts or adheres to the scaffold composition or device.

Scaffold compositions of the present invention contain an externalsurface. Alternatively, or in addition, scaffold compositions contain aninternal surface. External or internal surfaces of the scaffoldcomposition are solid or porous. Pore size is less than about 10 nm, inthe range of about 10 nm-20 μm in diameter, or greater than about 20 μm.A scaffold composition with multiple internal surfaces optionallycomprises one or more compartments.

Devices of the present invention are administered by intramuscularinjection. Alternatively, or in addition, scaffold compositions anddevices are administered by intraperitoneal injection, or endoscopicdelivery or other minimally invasive delivery approach, or surgicallyimplanted. Preferably, the loaded gel composition is injected as a bolususing a standard syringe and injection needle at or near the targetangiogenesis site, or it can be surgically implanted or delivered via acatheter.

The devices and methods of the invention provide a solution to severalproblems associated with previous angiogenesis-inducing protocols. Thebioactive composition is incorporated into or coated onto the scaffoldcomposition. The scaffold composition and/or bioactive compositiontemporally and spatially (directionally) controls release of one or moreadditional bioactive compositions.

This device includes a scaffold composition which incorporates into oris coated with a bioactive composition; the device releases one or morebioactive compositions comprised of pro-angiogenic factors and signalingmolecules to stimulate local vascular growth. Release of the bioactivecomposition is regulated spatially and temporally. Depending on theapplication for which the device is designed, the device regulatesrelease through the physical or chemical characteristics of the scaffolditself. For example, the scaffold composition is differentiallypermeable, allowing release only in certain physical areas of thescaffold. The permeability of the scaffold composition is regulated, forexample, by selecting or engineering a material for greater or smallerpore size, density, polymer cross-linking, stiffness, toughness,ductility, or viscoelascticity.

The scaffold composition contains physical channels or paths throughwhich bioactive compositions can move more easily towards a targetedarea of release of the device or of a compartment within the device. Thescaffold composition is optionally organized into compartments orlayers, each with a different permeability, so that the time requiredfor a bioactive composition to move through the device is precisely andpredictably controlled. Release is also regulated by the degradation,de- or re-hydration, oxygenation, chemical or pH alteration, or ongoingself-assembly of the scaffold composition. These processes are driven bydiffusion or catalyzed by enzymes or other reactive chemicals.

Alternatively, or in addition, release is regulated by a bioactivecomposition. By varying the concentration of extracellular matrixcomponents, adhesion molecules and other bioactive compounds indifferent areas of the device, including agents with means to createpores and enzymatically digest the scaffold composition, the bioactivecomposition has means to control the rate at which other elements of thesame bioactive composition or additional bioactive compositions escape,or are released from the scaffold composition or device. The devicecontrols and directs the flow of bioactive compositions or elementsthrough its structure.

Chemical affinities are used to channel bioactive compositions towards aspecific area of release. By varying the density and mixture of thosebioactive substances, the device controls the timing of the combinationand release of elements of bioactive compositions or multiple bioactivecompositions. In one embodiment, components of a bioactive compositionor two compositions are separated initially, but combined when allowedto flow through channels in the scaffold composition towards an area ofrelease. The density and mixture of these bioactive substances iscontrolled by initial doping levels or concentration gradient of thesubstance, by embedding the bioactive substances in scaffold materialwith a known leaching rate, by release as the scaffold materialdegrades, by diffusion from an area of concentration, by interaction ofprecursor chemicals diffusing into an area, or by production/excretionof compositions by neighboring cells.

Cells in close physical proximity to the scaffold compositions anddevices of the present invention, or those in physical contact with thescaffold composition and devices, secrete enzymes that affect the one ormore features of the scaffold composition. Neighboring or juxtaposedcells residing within target tissues increase or decrease the structuralintegrity of the scaffold composition through directly (e.g., release ofenzymes) or indirectly (e.g. release of signals recruiting cells to thescaffold compositions that affect structural integrity). Neighboring orjuxtaposed cells produce factors that increase or decrease the rigidity,increase or decrease the porosity, increase or decrease the potentialfor or rate of degradation, increase or decrease the adhesion ormobility, or increase or decrease the immunogenicity of the scaffoldcomposition or device. Alternatively, the scaffold composition iscomprised of materials that are unaffected by enzymatic activity and areunaffected by cellular secretions from local tissues.

The physical or chemical structure of the scaffold also regulates thediffusion of bioactive agents through the device. The release profilesof multiple bioactive compositions are made distinct from each other byadjusting the properties and formulation of delivery vehicle. Forexample, the small-molecule-weight signaling molecule is firstincorporated into microspheres followed by incorporation into alginatehydrogel, so that the signaling molecules have a more delayed releasecompared to the growth factors. The pore size, oxidization degree,molecular weight distribution of the alginate gel are varied to controlthe release rate of incorporated growth factors.

The bioactive composition includes one or more compounds that regulatecell function and/or behavior. The bioactive composition is covalentlylinked to the scaffold composition or non-covalently associated with thescaffold. For example, the bioactive composition is an extracellularmatrix (ECM) component that is chemically crosslinked to the scaffoldcomposition. Regardless of the tissue of origin, ECM componentsgenerally include three general classes of macromolecules: collagens,proteoglycans/glycosaminoglycans (PG/GAG), and glycoproteins, e.g.,fibronectin (FN), laminin, and thrombospondin. ECM components associatewith molecules on the cell surface and mediate adhesion and/or motility.Preferably, the ECM component associated with the scaffold is aproteoglycan attachment peptide or cyclic peptide containing the aminoacid sequence arginine-glycine-aspartic acid (RGD). Proteoglycanattachment peptides are selected from the group consisting of G₄RGDSP(SEQ ID NO: 1), XBBXBX (SEQ ID NO: 2), PRRARV (SEQ ID NO: 3),YEKPGSPPREVVPRPRPGV (SEQ ID NO: 4), RPSLAKKQRFRHRNRKGYRSQRGHSRGR (SEQ IDNO: 5), and RIQNLLKITNLRIKFVK (SEQ ID NO: 6), and cell attachmentpeptides are selected from the group consisting of RGD, RGDS, LDV, REDV,RGDV, LRGDN (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8), YIGSR (SEQ ID NO: 9),PDSGR (SEQ ID NO: 10), RNIAEIIKDA (SEQ ID NO: 11), RGDT, DGEA, and VTXG.

Components of the ECM, e.g., FN, laminin, and collagen, interact withthe cell surface via the integrin family of receptors, a group ofdivalent cation-dependent cell surface glycoproteins that mediatecellular recognition and adhesion to components of the ECM and to othercells. Ligands recognized by integrins typically contain an RGD aminoacid sequence that is expressed in many ECM proteins. Exemplarymolecules that mediate cell adhesion and/or movement include FN,laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin,aggrecan, agrin, bone sialoprotein, cartilage matrix protein,fibrinogen, fibrin, fibulin, mucins, entactin, osteopontin, plasminogen,restrictin, serglycin, SPARC/osteonectin, versican, von WillebrandFactor, polysaccharide heparin sulfate, cell adhesion moleculesincluding connexins, selectins include collagen, RGD (Arg-Gly-Asp) andYIGSR (Tyr-Ile-Gly-Ser-Arg) peptides, glycosaminoglycans (GAGs),hyaluronic acid (HA), integrins, selectins, cadherins and members of theimmunoglobulin superfamily. Carbohydrate ligands of the ECM include thepolysaccharides hyaluronic acid, and chondroitin-6-sulfate.

The device optionally contains a second or third bioactivecomposition(s), e.g., a growth factor, differentiation factor, orsignaling molecule. For example, the device includes vascularendothelial growth factor (VEGF), hepatocyte growth factor (HGF), orfibroblast growth factor 2 (FGF2) or a combination thereof. Growthfactors used to promote angiogenesis, wound healing, and other aspectsof tissue regeneration are listed herein and are used alone or incombination to induce regeneration of bodily tissues by bioactivecompositions released from an implanted scaffold device.

The bioactive composition(s) described above is non-covalently linked tothe scaffold composition. Alternatively, or in addition, the bioactivecomposition is covalently associated with the scaffold. Noncovalentbonds are generally one to three orders of magnitude weaker thancovalent bonds permitting diffusion of the factor out of the scaffoldand into surrounding tissues. Noncovalent bonds include electrostatic,hydrogen, van der Waals, π aromatic, and hydrophobic. For example, agrowth factor such as VEGF is associated with the device by noncovalentbonds and exits the device following administration of the device to atarget site to promote angiogenesis within the target bodily tissue.

The polymer scaffold composition into or onto which the bioactivecomposition (growth factor, and/or regulatory molecule are loaded isbiocompatible. The composition is bio-degradable/erodable or resistantto breakdown in the body. Relatively permanent (degradation resistant)scaffold compositions include metals and some polymers such as silk.Preferably, the scaffold composition degrades at a predetermined ratebased on a physical parameter selected from the group consisting oftemperature, pH, hydration status, and porosity, the cross-link density,type, and chemistry or the susceptibility of main chain linkages todegradation or it degrades at a predetermined rate based on a ratio ofchemical polymers. For example, a high molecular weight polymercomprised of solely lactide degrades over a period of years, e.g., 1-2years, while a low molecular weight polymer comprised of a 50:50 mixtureof lactide and glycolide degrades in a matter of weeks, e.g., 1, 2, 3,4, 6, 10 weeks. A calcium cross-linked gels composed of high molecularweight, high guluronic acid alginate degrade over several months (1, 2,4, 6, 8, 10, 12 months) to years (1, 2, 5 years) in vivo, while a gelcomprised of low molecular weight alginate, and/or alginate that hasbeen partially oxidized, will degrade in a matter of weeks. A typicalvolume of alginate gel is 1 μL to 1 mL, with a degradation time rangingfrom 1 day to 6 weeks.

In one example, cells mediate degradation of the scaffold matrix, i.e.,the scaffold composition is enzymatically digested by a compositionelicited by a neighboring cell, and the release of the bioactivecomposition is dependent upon the rate of enzymatic digestion of thescaffold. In this case, polymer main chains or cross-links containcompositions, e.g., oligopeptides, which are substrates for collagenaseor plasmin, or other enzymes produced by cells adjacent to the scaffold.

Exemplary scaffold compositions include polylactic acid, polyglycolicacid, PLGA polymers, alginates and alginate derivatives, gelatin,collagen, fibrin, hyaluronic acid, laminin rich gels, agarose, naturaland synthetic polysaccharides, polyamino acids, polypeptides,polyesters, polyanhydrides, polyphosphazines, poly(vinyl alcohols),poly(alkylene oxides), poly(allylamines)(PAM), poly(acrylates), modifiedstyrene polymers, pluronic polyols, polyoxamers, poly(uronic acids),poly(vinylpyrrolidone) and copolymers or graft copolymers of any of theabove. One preferred scaffold composition includes alginate gels.

Porosity of the scaffold composition influences release of one or morebioactive compositions from the device. Pores are nanoporous,microporous, or macroporous. For example, the diameter of nanopores areless than about 10 nm; micropore are in the range of about 100 nm-20 μmin diameter; and, macropores are greater than about 20 μm (preferablygreater than about 100 μm and even more preferably greater than about400 μm). In one example, the scaffold is macroporous with aligned poresof about 400-500 μm in diameter.

In one preferred embodiment of the invention, one or more of thebioactive compositions contains an element with means to chemicallyinduce pores in the scaffold composition that are nanoporous,microporous, or macroporous in size. The abundance of this element andthe presence or absence of other elements that augment or inhibit theactivity of this pore-making element determine pore size and density,and consequently, the rate at which one or more bioactive compositionspassively escape or are actively released from the scaffold composition.Exemplary elements with means to chemically induce pores in the scaffoldcomposition include, but are not limited to, potassium salts, calciumsalts, magnesium salts, amino acids, week acids, carbohydrates,potassium bitartrate, creatine, aspargine, glutamine, aspartic acid,glutamic acid, leucin, neroleucine, inosine, isoleucine, magnesiumcitrate, magnesium phosphate, magnesium carbonate, magnesium hydroxide,and magnesium oxide. Pore-forming elements are also enzymes incorporatedinto or coated onto the scaffold composition. One or more of theseelements are initially contained within separate compartments of thescaffold composition and later combined by having one or more of theseelements flow through common channels or paths in the scaffoldcomposition. These elements individually have means to induce poreformation in the scaffold composition. Alternatively, these elements arecombined, and the resulting combination has means to induce poreformation in the scaffold composition.

The devices are manufactured in their entirety in the absence of cellsor can be assembled around or in contact with cells (the material isgelled or assembled around cells in vitro or in vivo in the presence ofcells and tissues). In one embodiment of the invention, the scaffoldcomposition material with one or more bioactive compositionsincorporated, is injected into a target tissue where local blood flow isrestricted or would healing is impaired.

The device is manufactured in one stage comprising one layer orcompartment. Alternatively, the device is manufactured in two or more(3, 4, 5, 6, . . . 10 or more) stages in which one layer or compartmentis made and infused or coated with a bioactive composition followed bythe construction of a second, third, fourth or more layers, which are inturn infused or coated with a bioactive composition in sequence. Eachlayer or compartment is identical to the others or distinguished fromone another by the elements comprising the bioactive compositionincorporated into or coated onto them as well as distinct chemical,physical and biological properties.

A method of making a scaffold is carried out by providing a scaffoldcomposition and covalently linking or noncovalently associating thescaffold composition with a first bioactive composition. The scaffoldcomposition is also contacted with a second bioactive composition. Thesecond bioactive composition is non-covalently associated with thescaffold composition to yield a doped (loaded) scaffold, i.e., ascaffold composition that includes one or more bioactive substances. Thecontacting steps are optionally repeated to yield a plurality of dopedscaffolds, e.g., each of the contacting steps is characterized by adifferent amount of the second bioactive composition to yield a gradientof the second bioactive composition in the scaffold device. Rather thanaltering the amount of composition, subsequent contacting steps involvea different bioactive composition, i.e., a third, fourth, fifth, sixth .. . , composition or mixture of compositions, that is distinguished fromthe prior compositions or mixtures of prior doping steps by thestructure or chemical formula of the factor(s). The method optionallyinvolves adhering individual niches, layers, or components to oneanother and/or insertion of semi-permeable, permeable, or nonpermeablemembranes within or at one or more boundaries of the device to furthercontrol/regulate locomotion of cells or bioactive compositions.

Therapeutic applications of the device include vascular tissuegeneration, regeneration and repair. A mammalian tissue is contactedwith the device. The scaffold composition and/or the bioactivecomposition spatially or directionally regulates release of a bioactivecomposition with means to promote angiogenesis in local tissues such asvascular, muscle, gastrointestinal, e.g., bowel, cardiac, brain, kidney,bone, nerve both central and peripheral nervous system (CNS and PNS) orany tissue characterized by a shortage of oxygen or nutrients or inwhich the vasculature is damaged, absent, or functionally impaired.

A method of inducing local angiogenesis in a target tissue is carriedout by administering to a mammal a device containing a scaffoldcomposition and a bioactive composition incorporated therein or thereon.The scaffold composition and/or bioactive composition induces release ofpro-angiogenic factors and signaling molecules from the device into thelocal tissue environment. The release of these angiogenic factors iscontrolled spatially and temporally. Release can be sustained at asteady rate/dosage for a desired period of time, e.g., minutes; 0.2.0.5, 1, 2, 4, 6, 12, 24 hours; 2, 4, 6, days; weeks (1-4), months (2, 4,6, 8, 10, 12) or years, during which the cells are exposed to structuralelements and bioactive compositions that lead to improved vasculargrowth.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. All references cited herein are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the effect of VEGF on sprouting ratio. Anin vitro model was used to test the significance of a controlled localconcentration of VEGF.

FIG. 2 is a bar graph showing the effect of VEGF and GSI on sproutingratio. In vitro model was used to test the significance of a combinationof DAPT and VEGF.

FIG. 3 is a bar graph showing the effect of different amounts and ratiosof amounts of VEGF and GSI on sprouting ratio. In vitro modelestablishing the significance of a distinct presentation of VEGF andDAPT.

FIGS. 4A-C are a series of line graphs showing the effect of VEGF and aGSI (DAPT) on angiogenesis. 4A, In vitro release profiles of VEGF andDAPT from injected alginate hydrogel system; 4B and 4C, In vivo model totest the effect of controlled presentation of VEGF and DAPT to recoverblood flow in an ischemia situation. Blood flow recovery subject tohindlimb ligation. gel: represents the use of the alginate as thedelivery vehicle. im: intramuscular injection. ip: intraperitonealinjection.

FIGS. 5A-B are bar graphs showing the effect of VEGF and DAPT on bloodcapillary density. An in vivo model was used to test the effect ofcontrolled presentation of VEGF and DAPT on newly formed blood vesseldensity in an ischemia situation. +: use of the substance. −: lack ofthe substance. gel: alginate gel. im: intramuscular injection. ip:intraperitoneal injection.

FIG. 6 is a series of photomicrographs showing the effect of DAPT ongastrointestinal tissue. In vivo model was used to test the effect ofDAPT delivered from alginate gel system and from intraperitonealinjection on the cells in small intestines. gel: alginate gel. ip:intraperitoneal injection. H&E, alcian blue, Ki67 and HES-1 are the fourdifferent staining methods to characterize the crypt cells in smallintestines. Ki-67 was used to stain the proliferative cells. Loss ofNotch signaling can alter the proliferation rate of crypt cells. HES-1staining was to examine the expression of a known Notch target gene incrypts. Alcian blue staining was to examine the deposition ofglycosaminoglycan molecules. Loss of Notch signaling can result in moredeposition of glycosaminoglycan molecules. Expression of HES-1 (hairyand enhancer of split 1), a member of basic helix-loop-helix family oftranscription factors and a known Notch target gene in crypts wasexamined. Loss of Notch signaling can alter the proliferation rate ofcrypt cells, as shown by Ki-67 staining the proliferative cells. Inaddition, Notch inhibition has been reported to alter the balancebetween proliferative crypt cells and goblet cells, resulting in moredeposition of glycosaminoglycan molecules, as characterized by alcianblue staining Notch inhibition can also result in a significantalteration of the morphology of the small intestine as compared tocontrols, as demonstrated by hematoxylin and eosin (H&E) staining.

FIG. 7A is a bar graph showing the effect of VEGF, DAPT, and thecombination on blood capillary density.

FIG. 7B is a line graph showing the effect of VEGF, DAPT, and thecombination on blood flow.

FIG. 8A is a bar graph showing the effect of VEGF, PDGF, and thecombination on blood capillary density.

FIG. 8B is a line graph showing the effect of VEGF, PDGF, and thecombination on blood flow.

FIG. 9A is a bar graph showing the effect of VEGF, DAPT, PDGF, andcombinations thereof on blood capillary density.

FIG. 9B is a line graph showing the effect of VEGF, DAPT, PDGF, andcombinations thereof on blood flow.

FIG. 10A is series of photomicrographs showing the effect of VEGF, DAPT,PDGF, and combinations thereof on maturation of newly formed bloodvessels in the local muscle tissues around the ischemic site.

FIGS. 10B-C are bar graphs showing the effect of VEGF, DAPT, PDGF, andcombinations thereof on maturation of newly formed blood vessels asmeasured by density of smooth muscle actin (SMA) positive vessels perunit area (FIG. 10B) and the percentage of SMA positive vessels (FIG.10C).

DETAILED DESCRIPTION OF THE INVENTION

The use of compounds that can modulate the signaling of growth factors,e.g., gamma-secretase inhibitors (GSI), to promote angiogenesis, hasbeen proposed (US 2006/0264380 A1). However, these previous studies didnot indicate a need to provide the compounds over particulartime-frames, nor do they provide any method to achieve a sustainedpresence of the compounds. Previous studies mention the use of acombination of pro-angiogenic factors together with GSI. Functionally, abolus injection of multiple pro-angiogenesis compounds simultaneouslycannot control the presentation of each individual compound separately,as they may each need to have a distinct presentation. More importantly,single or multiple injections that introduce drugs into systemcirculation represent a sub-optimal method to reach the therapeuticlevel at the specific tissue of interest, and can result in highconcentrations of drug accumulation at distant organs or tissues, whichmay lead to various side effects.

The current invention provides a method to control the localpresentations of pro-angiogenic growth factors and signaling moleculesthat are together used to achieve angiogenesis at the tissue or organ ofinterest. The spatial and temporal presentation of delivered growthfactors and signaling molecules can be controlled separately byfine-tuning the physical and chemical properties of the polymer deliverymaterial.

Specifically, this invention will be especially useful for developingtreatments for chronic ischemia in coronary or peripheral artery diseasefor diabetics, or wound healing in diabetes ulcers, as the impairedendothelium in these patients normally have a reduced response toregular pro-angiogenesis factors, thus a continuous activation by thesustained and distinct presentation of growth factors and signalingmolecules may be critical. This is currently not achievable by othermethods without inducing possible side-effects.

Angiogenesis refers to a process of new blood vessel formation. Patientssuffering from coronary arterial disease (CAD) and peripheral arterialdisease (PAD) can be treated by promoting angiogenesis in the tissuelacking sufficient blood blow. To deliver compounds to induceangiogenesis is therefore a promising therapeutic approach. Many growthfactors, (e.g. vascular endothelial growth factor (VEGF), play a vitalrole in inducing angiogenesis by mediating the proliferation, migrationand differentiation of endothelial cells. Molecules that can modulatethe signaling pathways of growth factors, such as Notch inhibitors(e.g., gamma secretase inhibitors (GSI)), can also be used to augmentthe angiogenesis process. Therefore, delivering growth factors togetherwith molecules mediating signaling pathways may have a beneficialeffect.

Current delivery approaches mainly rely on injection offactors/molecules alone, i.e., in the absence of a scaffold composition.However, single injection is insufficient for circumstances where growthfactors and signaling molecules need to be present over longtime-frames, as they both have a short half-life. In addition, bolusinjection of multiple compounds simultaneously cannot control theexistence of each individual compound separately, if they each need tohave a distinct presentation. More importantly, single or multipleinjections that introduce drugs into system circulation are sub-optimalto reach the therapeutic level at the specific tissue of interest, andcan result in high concentrations at distant organs or tissues, whichmay lead to various side effects.

The current invention utilizes an injectable biocompatible polymermaterial system incorporating pro-angiogenic growth factors, togetherwith molecules that modulate the signaling pathway, to achieveangiogenesis at the tissue or organ of interest. The spatial andtemporal presentation of delivered growth factor and signaling moleculescan be controlled separately by fine-tuning the physical and chemicalproperties of the polymer material.

Angiogenesis

Angiogenesis is a physiological process wherein new blood vessels ariseor extend from pre-existing vessels. The present invention encompassesmethods of promoting blood vessel growth from existing vessels, as wellas spontaneous blood vessel growth (also referred to as vasculogenesis)and arteriogenesis (collateral vessel formation). The term“angiogenesis” is meant to encompass all three methods of blood vesselformation named supra.

Angiogenesis is a normal process in growth and development, as well asin wound healing. Compositions and methods of the present inventioninduce or augment endogenous mechanisms for regulating angiogenesisand/or wound healing. Alternatively, or in addition, compositions andmethods of the present invention introduce exogenous mechanisms forinducing or regulating angiogenesis and/or wound healing that do notnormally occur in a target tissue. Furthermore, compositions and methodsof the present invention induce, regulate, augment, or replacemechanisms for inducing or regulating angiogenesis and/or wound healingthat are insufficient, aberrant, or incomplete compared to theendogenous mechanism due to genetic mutation, disease, infection, drugtreatment, medical condition, or tissue transplant procedure.

Angiogenesis is also a fundamental step in the transition of neoplastictumors from a dormant, or benign, state to a malignant, cancerous, ormetastatic, state. Compositions and methods of the present inventiondisperse angiogenic factors and signaling molecules within a localtissue environment and do not allow for systemic administration ordiffusion of bioactive compositions. This ability of the devices of thepresent invention to contain angiogenic factors to a confined targetregion demonstrates a significant clinical advantage over previousmethods of delivery that are systemic in nature.

Controlled Release of Factors to Promote Angiogenesis

The release profiles of bioactive substances from scaffold devices iscontrolled by both factor diffusion and polymer degradation, the dose ofthe factor loaded in the system, and the composition of the polymer.Similarly, the range of action (tissue distribution) and duration ofaction, or spatiotemporal gradients of the released factors areregulated by these variables. The diffusion and degradation of thefactors in the tissue of interest is optionally regulated by chemicallymodifying the factors (e.g., PEGylating growth factors). In both cases,the time frame of release determines the time over which effectiveangiogenesis by the device is desired.

In the current system, the degradation rate of alginate, the polymercarrier, is controlled by its composition (for example, content ofguluronic acid of alginate molecules, differential molecular weightdistribution of alginate), physical and chemical treatment (e.g.,irradiation or oxidization), and the degree of crosslinking which iscontrolled by the choice and the amount of the crosslinking agents (fore.g., ionic crosslinker or covalent crosslinker). More specifically,increasing the content of guluronic acid of alginate molecules willincrease cross-linking and slow degradation, decreasing molecular weightwill speed up degradation, irradiation will decrease molecular weightand thus increase the degradation rate, oxidization will increase thedegradation rate, increasing the amount of crosslinker will slow thedegradation rate, and different crosslinker molecules may result indifferential crosslinking degree and affect the degradation rate.

The doses of the factors loaded in the alginate carrier are altered toachieve effective doses at a desired tissue site, e.g., from 1 to 10microgram for VEGF₁₆₅, 1 to 10 microgram for PDGF-BB (dimericglycoprotein composed of two B (-BB) chains), and 0.01 to 1 microgramfor DAPT, for a mouse with an average weight of 10-50 g. An effectivedosage amount or ratio of amounts is one that induces and promotesangiogenesis at the target tissue or organ site. The optimal dose is 3to 10 microgram for VEGF₁₆₅, 3 to 10 microgram for PDGF-BB, 0.05 to 1microgram for DAPT in the presence of VEGF at a dose of 1 to 10microgram, and 0.05 to 1 microgram for DAPT in the presence of PDGF-BBat a dose of 1 to 10 microgram, for a mouse with an average weight of10-50 g. The amounts and ratio of amounts scale up proportionately forhumans.

The relative ratio of VEGF, PDGF and the Notch signaling molecule (e.g.,DAPT) is critical in determining the final outcomes of angiogenesis.There exists an effective range and optimal value of the relative ratiobetween the amount of VEGF and DAPT, the amount of PDGF and DAPT, andthe amount of VEGF, PDGF and DAPT. The effective range of the relativeratio (by mole) is 1:1 to 1:200 for VEGF₁₆₅ to DAPT, 1:1 to 1:200 forPDGF-BB to DAPT, and 1:0.1:1 to 1:10:200 for VEGF₁₆₅, PDGF-BB and DAPT,in the alginate polymer system. The optimal relative ratio (by mole) is1:31 for VEGF₁₆₅ to DAPT, 1.8:31 for PDGF to DAPT, and 1:1.8:31 forVEGF₁₆₅:PDGF-BB:DAPT. The optimal ratio may vary depending on thespecific delivery systems to be used, the species model (e.g., rat,rabbit, pig, dog, human and etc), and any change of the composition andthe release kinetics of each of these factors.

Arteriosclerosis

Arteriosclerosis, or arterial disease, is a general term used todescribe the thickening and hardening of the arteries. One particularkind of arteriosclerosis that contributes to heart disease isatherosclerosis. Atherosclerosis is a progressive disease that ischaracterized by a buildup of plaque within the arteries that maypartially, or totally, block blood flow through an artery. Plaque isformed from fatty substances, cholesterol, cellular waste, calcium, andfibrin. Atherosclerosis generally results in ischemia, or restriction ofthe blood supply, for the tissues supplied by the blood carried in theblocked artery.

Oxygen Supply and Deprivation

Because oxygen is mainly bound to hemoglobin in red blood cells,insufficient blood supply causes tissue to become hypoxic, or, in moresevere situations, when no oxygen is supplied, anoxic. Oxygendeprivation can cause necrosis, or cell death. In very aerobic tissuessuch as heart and brain, at body temperature, necrosis due to ischemiabecomes irreversible in 3-4 hours. Complete oxygen deprivation to organssuch as the heart and brain for greater than 20 minutes causesirreversible damage.

Ischemia is a consequence of heart diseases, transient ischemic attacks,cerebrovascular accidents, ruptured arteriovenous malformations, andperipheral artery occlusive disease. The heart, kidneys, and brain arethe most sensitive organs to inadequate blood supply. Stroke, aneurism,hemorrhage, and traumatic injury of the brain commonly result inischemic conditions. Ischemia in brain tissue induces the ischemiccascade, in which proteolytic enzymes, reactive oxygen species, andother harmful chemicals damage and may ultimately kill brain tissue.Similarly, artery disease and blockages can occlude blood flow to theheart inducing ischemia and death of heart muscle tissue. A macroscopicregion of necrotic cells is called an infarction. Heart attacks lead tosignificant cell death from prolonged oxygen deprivation, also referredto as myocardial infarction.

Restoration of blood flow after a period of ischemia may cause moredamage than the ischemia. Reintroduction of oxygen causes a greaterproduction of damaging free radicals, resulting in reperfusion injuryand accelerated necrosis.

Coronary Arterial Disease

Patients diagnosed with Coronary Arterial Disease (CAD, also calledcoronary heart disease, coronary artery disease, ischaemic heartdisease, and atherosclerotic heart disease) results from theaccumulation of atheromatous plaques within the walls of the arteriesthat supply the myocardium (heart muscle) with oxygen and nutrients. CADencompasses a wide spectrum of patients with varying disease severityand prognosis. Patients with mild CAD and the best prognoses areasymptomatic. Mild CAD individuals have atheromatous streaks within thewalls of their coronary arteries that do not obstruct blood flow and thelumen of their coronary artery is normal in calibre (as assessed bycoronary angiogram). As an individual progresses along this spectrumtoward more severe phenotypes, the atheromatous streaks along thecoronary walls increase in thickness. Atheromatous plaques begin to forminitially and expand into the walls of the artery but, ultimately,expand into the luman of the vessel where they will begin to restrictblood flow.

Once the plaques obstruct more than 70% of the diameter of the vessellumen, the individual develops symptoms of obstructive coronary arterydisease and is diagnosed with ischemic heart disease. The first symptomsof ischemic heart disease are often exertional angina (chest pain) ordecreased exercise tolerance. Angina that occurs regularly withactivity, upon awakening, or at other predictable times is termed stableangina. Angina that changes in intensity, character or frequency istermed unstable. Unstable angina may precede myocardial infarction.

The degree of severity of CAD can progress to near-complete or completeblockage of the coronary artery. At this end of the spectrum, mostindividuals experience one or more heart attacks (myocardialinfarctions) and all experience chronic ischemia. If the blood flow tothe heart tissue is restored to any degree, ischemic tissue is capableof a partial or full recovery depending upon the degree of blood flowrestoration. Tissue that has suffered from an infarction is dead, andthe damage is irreversible.

Peripheral Arterial Disease

Peripheral Arterial Disease (PAD, also called peripheral arteryocclusive disease (PAOD), peripheral vascular disease, and peripheralartery disease) is caused by the obstruction of large peripheralarteries, which can result from atherosclerosis or inflammatoryprocesses and can lead to a narrowing of the artery (stenosis) orobstruction of the artery by thrombus (obstruction by blood clot) orembolism (obstruction by object carried in blood stream from alternatelocation). PAD/PAOD results in ischemia that is either acute (rapidonset, short duration) or chronic (long-term). Exemplary symptoms ofPAD/PAOD include, but are not limited to, claudication (pain, weakness,or cramping in muscles due to decreased blood flow); sores, wounds, orulcers that heal slowly or incompletely; change in color (blueness,paleness) or cooling compared to other limbs; diminished hair or nailgrowth on affected limbs compared to unaffected limbs.

PAD/PAOD occurrence is often associated with or caused by smoking,diabetes mellitus, dyslipidemia (e.g. elevated cholesterol, includingtotal cholesterol, LDL cholesterol, and triglyceride levels),hypertension, increased or decreased levels of inflammatory mediators(for example, C-reactive protein, homocysteine, and fibrinogen), aging(especially individuals over 50), racial background (especiallyprevalent among African-American individuals), gender (more frequentlyseen in males), obesity, or individuals with personal histories ofvascular disease, heart attack, or stroke. The present inventionencompasses methods of administering compositions, scaffolds, anddevices to all individuals listed supra, for the purposes of repairingor replenishing blood supply to blood- and oxygen-deprived tissues.

PAD/PAOD is diagnosed using a number of tests. The initial test is anankle brachial pressure index (ABPI/ABI) which measures the fall inblood pressure in the arteries supplying the legs. A reduced ABPI,quantitatively, a score of less than 0.9, suggests a diagnosis ofPAD/PAOD. Moderate PAD/PAOD is diagnosed with a reduced ABPI score ofless than 0.8. Severe PAD/PAOD is diagnosed with a reduced ABPI score ofless than 0.5. However, conditions other than PAD/PAOD can result inreduced ABPI scores of less than 0.9. Thus, additional tests areperformed to confirm a diagnosis of PAD/PAOD. A secondary examinationusually comprises a lower limb Doppler ultrasound examination of thefemoral artery. Alternatively, or in addition, imaging examinations canbe performed by angiography using art-recognized standard methods.Furthermore, a multi-slice computerized tomography (CT) scan is used todirectly image the arterial system.

PAD/PAOD severity is divided in the Fontaine stages (Fontaine R, Kim M,Kieny R (1954). Helvetica Chirurgica Acta, Basel 21 (5/6):499-533): mildpain while walking (“claudication”)(stage I); severe pain on walkingrelatively shorter distances (intermittent claudication)(stage II); painwhile resting (stage III); loss of sensation to the lower part of theextremity (stage IV); tissue loss (gangrene)(stage V).

Angiogenic Bioactive Compositions

Compositions and methods of the present invention comprise growthfactors and signaling molecules that induce, regulate, or augmentangiogenesis. Bioactive compositions of the present invention compriseone or more growth factors or signaling molecules incorporated into orcoated onto the scaffold composition. Exemplary growth factors andsignaling molecules encompassed by the present invention include, butare not limited to, vascular endothelial growth factor (VEGF (A-F)),fibroblast growth factors (acidic and basic FGF 1-10),granulocyte-macrophage colony-stimulating factor (GM-CSF), insulin,insulin growth factor or insulin-like growth factor (IGF), insulingrowth factor binding protein (IGFBP), placenta growth factor (PIGF),angiopoietin (Ang1 and Ang2), platelet-derived growth factor (PDGF),hepatocyte growth factor (HGF), transforming growth factor (TGF-α,TGF-β, isoforms 1-3), platelet-endothelial cell adhesion molecule-1(PECAM-1), vascular endothelial cadherin (VE-cadherin), nitric oxide(NO), chemokine (C-X-C motif) ligand 10 (CXCL10) or IP-10, interleukin-8(IL-8), hypoxia inducible factor (HIF), monocyte chemotactic protein(MCP), vascular cell adhesion molecule (VCAM), ephrin ligands (includingEphrin-B2 and -B4); Transcription factors such as HIF-1α, HIF-10 andHIF-2α, Ets-1, Hex, Vezf1, Hox, GATA, LKLF, COUP-TFII, Hox, MEF2, Braf,Prx-1, Prx-2, CRP2/SmLIM and GATA family members, basic helix-loop-helixfactors and their inhibitors of differentiation; and regulatorymolecules include enzymes (matrix metalloproteinase (MMP), tissueplasminogen activator (PLAT or tPA), cyclooxygenase (COX), angiogenin),molecules regulating Notch signaling which consists of monoclonalantibodies to Notch ligands and receptors, RNA interference, antisenseNotch, receptor and mastermind-like 1 (MAML1) decoys, beta andgamma-secretase inhibitors (GSI), or any other molecules that canactivate or inhibit Notch signaling.

Vascular Endothelial Growth Factor (VEGF, also known as VEGF-A)

The term “VEGF” broadly encompasses two families of proteins that resultfrom the alternate splicing of a single gene, VEGF, composed of 8 exons.The alternate splice sites reside in the exons 6, 7, and 8. However, thealternate splice site in the terminal exon 8 is functionally important.One family of proteins arise from the proximal splice site and aredenoted (VEGF_(xxx)). Proteins produced by alternate splicing at thisproximal location are PRO-angiogenic and are expressed conditionally(for instance, when tissues are hypoxic and secreted signals induceangiogenesis). The other family of proteins arise from the distal splicesite and are denoted (VEGF_(xxx)b). Proteins produced by alternatesplicing at this distal location are ANTI-angiogenic and are expressedin healthy tissues under normal conditions.

VEGF exons 6 and 7 contain splice sites (result in the inclusion orexclusion of exons 6 and 7) that affect heparin binding affinity andamino acid number. Humans comprise VEGF₁₂₁, VEGF₁₂₁b, VEGF₁₄₅, VEGF₁₆₅,VEGF₁₆₅b, VEGF₁₈₉, and VEGF₂₀₆. Heparin binding affinity, interactionswith heparin surface proteoglycans (HSPGs) and neuropilin co-receptorson the cell surface mediated by amino acid sequences in exons 6 and 7enhance the ability of VEGF variants to activate VEGF signalingreceptors (VEGFRs).

Endogenous VEGF splice variants are released from cells as glycosylateddisulfide-bonded dimers. Structurally VEGF belongs to the PDGF family ofcysteine-knot growth factors comprising Placenta growth factor (PlGF),VEGF-B, VEGF-C and VEGF-D (the VEGF sub-family of growth factors). VEGFis sometimes referred to as VEGF-A to differentiate it from theserelated growth factors. The term “VEGF” used herein to describe thepresent invention is meant to refer to VEGF-A.

Members of the VEGF family stimulate cellular responses by binding tocell-surface tyrosine kinase receptors (the VEGFRs). VEGF-A binds toVEGFR-1 (also known as Flt-1) and VEGFR-2 (also known as KDR/Flk-1).VEGFR-2 is the predominant receptor for VEGF-A mediating almost all ofthe known cellular responses to this growth factor. The function ofVEGFR-1 is unclear, although it is thought to modulate VEGFR-2signaling. VEGFR-1 may also sequester VEGF from VEGFR-2 binding (whichmay be important during development).

Compositions, methods, and devices of the present invention comprise allVEGF polypeptides generated from alternative splicing including pro- andanti-angiogenic forms. Devices of the present invention administered toa subject contain only pro-angiogenic VEGF polypeptide splice forms.Alternatively, or in addition, devices of the present inventionadministered to a subject contain a mixture of pro- and anti-angiogenicVEGF polypeptide splice forms. Pro- and anti-angiogenic VEGF polypeptidesplice forms are released by the scaffold composition of the devicesimultaneously or sequentially. For example, the opposing splice formsare released together in order to achieve a precise level ofstimulation. Alternatively, the opposing splice forms are releasedsequentially to stimulate angiogenesis and subsequently attenuate thesignal when the desired result has been achieved. In another embodiment,devices comprising pro-angiogenic VEGF polypeptide splice forms areplaced at the target tissue site while devices comprisinganti-angiogenic VEGF polypeptide splice forms are placed in surroundingtissues in order to prevent pro-angiogenic signals from disseminatinginto and stimulating non-target tissue.

Exemplary VEGF polypeptide splice forms comprised by the compositions,methods, and devices of the present invention include, but are notlimited to, the polypeptides described by the following sequences andSED ID NOs. VEGF polypeptide splice forms are released fromcompositions, scaffolds, or devices of the present invention as naked,or glycosylated polypeptides. Alternatively, or in addition, VEGFpolypeptide splice forms are monomers or disulfide-bonded dimers. In apreferred embodiment, VEGF polypeptide splice forms are released intotarget tissues from compositions, scaffolds, and/or devices of thepresent invention as glycosylated disulfide-bonded dimers.

Human VEGF₁₄₈ is encoded by the following amino acid sequence (NCBIAccession No. NP_(—)001020540 and SEQ ID NO: 12):

1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg 61csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt 121geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem 301sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt ckcsckntds rckm

Human VEGF₁₆₅ is encoded by the following amino acid sequence (NCBIAccession No. NP_(—)001020539 and SEQ ID NO: 13):

1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg 61csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt 121geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem 301sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt ckcsckntds rckarqleln 361ertcrcdkpr r

Human VEGF₁₆₅b is encoded by the following amino acid sequence (NCBIAccession No. NP_(—)001028928 and SEQ ID NO: 14):

1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg 61csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt 121geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem 301sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt ckcsckntds rckarqleln 361ertcrsltrk d

Human VEGF₁₈₃ is encoded by the following amino acid sequence (NCBIAccession No. NP_(—)001020538 and SEQ ID NO: 15):

1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg 61csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt 121geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem 301sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksrp cgpcserrkh lfvqdpqtck 361csckntdsrc karqlelner tcrcdkprr

Human VEGF₁₈₉ is encoded by the following amino acid sequence (NCBIAccession No. NP_(—)003367 and SEQ ID NO: 16):

1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg 61csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt 121geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd 241ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem 301sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvpcgpc serrkhlfvq 361dpqtckcsck ntdsrckarq lelnertcrc dkprr

Human VEGF₂₀₆ is encoded by the following amino acid sequence (NCBIAccession No. NP_(—)001020537 and SEQ ID NO: 17):

1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg 61csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt 121geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset 181mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd 241ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem 301sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvyvgar cclmpwslpg 361phpcgpcser rkhlfvqdpq tckcsckntd srckarqlel nertcrcdkp rrGamma-Secretase Inhibitors (GSI)

Notch is a cell-surface receptor that regulates cell fate decisionsthroughout development and under selected conditions in adult tissues.Notch signaling results in widely variable outcomes depending on thecells and signaling molecules involved. However, it is generally knownthat binding of Notch ligands of the Delta and Jagged families resultsin the proteolytic cleavage of Notch. The Notch protein is first cleavedin the extracellular domain and then subsequently cleaved in thetransmembrane domain. The second cleavage event is mediated byγ-secretase. Notch cleavage allows the intracellular domain of thereceptor (the Notch IntraCellular Domain, NICD) to translocate to thenucleus where it regulates transcription. Thus, γ-secretase is a Notchactivator.

Notch signaling is involved in angiogenesis and vascular remodeling.Moreover, Notch signaling regulates endothelial cell proliferation andmigration events necessary to form new blood vessels during angiogenesisin normal tissues as well as malignant tumors. Methods of the presentinvention are drawn towards inducing angiogenesis in normal tissues, notmalignant tissues. Furthermore, it is of great importance to avoidinducing a malignant state within a stable or benign tumor byintroducing pro-angiogenic factors in the absence of factors to limitNotch activation. In one preferred embodiment of the present invention,pro-angiogenic factors are released from compositions, scaffolds, ordevices, either simultaneously or sequentially, with notch-inhibitors,e.g. inhibitors of gamma-secretase (γ-secretase), to prevent stimulationof angiogenesis within neoplastic tissue.

Compositions, scaffolds, and devices of the present invention compriseall inhibitors of Notch activation to be released simultaneously orsequentially with pro-angiogenic factors. Inhibitors of Notch activityencompassed by the present invention block binding of one or moreligands to the Notch receptor. Alternatively, or in addition, inhibitorsof Notch activity present intracellular signal transduction from theNotch receptor or cleavage of the Notch receptor polypeptide. Notchinhibitors of the present invention comprise endogenous or exogenoussmall molecules, compounds, single- or double-stranded RNApolynucleotides, single- or double-stranded DNA polynucleotides,polypeptides, antibodies, intrabodies, natural or synthetic ligands,genetically-engineered ligands, and genetically-manipulated γ-secretaseproteins or fragments thereof. Exemplary inhibitors of Notch activationinclude, but are not limited to, monoclonal antibodies to Notch ligandsand receptors, RNA interference, antisense Notch, receptor andmastermind-like 1 (MAML1) decoys, beta and gamma-secretase inhibitors(GSI).

Gamma-secretase is an integral membrane protein that is one part of amulti-subunit protease complex that cleaves single-pass transmembraneproteins at residues within the transmembrane domain. The gammasecretase complex comprises four individual proteins: presenilin,nicastrin, APH-1 (anterior pharynx-defective 1), and PEN-2 (presenilinenhancer 2). A fifth protein, known as CD147, is a non-essentialregulator of the complex whose absence increases activity.

The proteins in the gamma secretase complex are heavily modified byproteolysis during assembly and maturation of the complex. Presenilin isan aspartyl protease comprising the catalytic subunit and is activatedby autocatalytic cleavage of to N- and C-terminal fragments. Nicastrinmaintains the stability of the assembled complex, regulatesintracellular protein trafficking, and recognizes substrates via bindingto the N-terminal ectodomain of the target protein. PEN-2 associateswith the complex via binding of a transmembrane domain of presenilin andstabilizes the complex after presenilin proteolysis. APH-1, which isrequired for proteolytic activity, binds to the complex via a conservedalpha helix interaction motif and aids in initiating assembly ofpremature components.

The present invention comprises one or more inhibitors of γ-secretasewhich target one or more proteins of this complex and inhibit one ormore functions of these proteins. Alternatively, or in addition,γ-secretase inhibitors of the present invention prevent assembly of theγ-secretase protease complex. Furthermore, contemplated γ-secretaseinhibitors of the present invention inhibit intracellular signaltransduction from an assembled γ-secretase protease complex.Contemplated γ-secretase inhibitors of the present invention bind one ormore proteins of the g-secretase complex and partially or entirely blockan activity or function. Exemplary γ-secretase inhibitors of the presentinvention decrease, prevent, or delay activation, as well as inactivateone or more protease components. Exemplary γ-secretase inhibitors of thepresent invention desensitize or down regulate the activity orexpression of one or more proteins of the γ-secretase complex. Exemplaryγ-secretase inhibitors of the present invention consist of, consistessentially of, or comprise endogenous or exogenous small molecules,compounds, single- or double-stranded RNA polynucleotides, single- ordouble-stranded DNA polynucleotides, polypeptides, antibodies,intrabodies, natural or synthetic ligands, genetically-engineeredligands, and genetically-manipulated γ-secretase proteins or fragmentsthereof.

Exemplary γ-secretase inhibitors of the present invention include, butare not limited to, DAFT andN-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester.

Cell-Mediated Enzymatic Scaffold Degradation

Cells secrete enzymes that degrade the material of the scaffold, therebycontrolling the rate at which cells exit the scaffold. Cells withinclose proximity to the implanted scaffold composition secrete enzymes,such as collagenases and plasmin, which degrade the polymer composition.This property is used in certain embodiments to control the release ofbioactive compositions into the local cellular environment. The rate ofrelease of bioactive composition may thus be regulated by controllingthe density and susceptibility to these enzymes of oligopeptides used aseither cross-links in the material or as components of the main chains.Certain materials are degraded in a preprogrammed manner independent ofcell action (e.g. hydrolytic degradation of poly(lactide-co glyolide) asa degradable scaffold. The scaffolds may be prepared such that thedegradation time may be controlled by using a mixture of degradablecomponents in proportions to achieve a desired degradation rate.Scaffold compositions are sensitive to degradation by materials secretedby the cells located immediately adjacent to the scaffold. One exampleof this is the use of metalloproteinase (MMP)-sensitive substrate in thescaffold matrix; bioactive composition is released when the adjacentcells have secreted sufficient MMP to begin degradation of the matrix.

Scaffold Compositions and Architecture

Components of the scaffolds are organized in a variety of geometricshapes (e.g., beads, pellets), niches, planar layers (e.g., thinsheets). For example, multicomponent scaffolds are constructed inconcentric layers each of which is characterized by different physicalqualities (% polymer, % crosslinking of polymer, chemical composition ofscaffold, pore size, porosity, and pore architecture, stiffness,toughness, ductility, viscoelasticity, and or composition of bioactivesubstances such as growth factors, homing/migration factors,differentiation factors. Each niche has a specific effect on a cellpopulation, e.g., promoting or inhibiting a specific cellular function,proliferation, differentiation, elaboration of secreted factors orenzymes, or migration.

Cells incubated in the scaffold are educated and induced to migrate outof the scaffold to directly affect a target tissue, e.g., and injuredtissue site. For example, stromal vascular cells and smooth muscle cellsare useful in sheet-like structures are used for repair of vessel-likestructures such as blood vessels or layers of the body cavity. Forexample, such structures are used to repair abdominal wall injuries ordefects such as gastroschisis. Similarly, sheet-like scaffolds seededwith dermal stem cells and/or keratinocytes are used in bandages orwound dressings for regeneration of dermal tissue. The device is placedor transplanted on or next to a target tissue, in a protected locationin the body, next to blood vessels, or outside the body as in the caseof an external wound dressing. Devices are introduced into or onto abodily tissue using a variety of known methods and tools, e.g., spoon,tweezers or graspers, hypodermic needle, endoscopic manipulator, endo-or trans-vascular-catheter, stereotaxic needle, snake device,organ-surface-crawling robot (United States Patent Application20050154376; Ota et al., 2006, Innovations 1:227-231), minimallyinvasive surgical devices, surgical implantation tools, and transdermalpatches. Devices can also be assembled in place, for example bysequentially injecting or inserting matrix materials. Scaffold devicesare optionally recharged with cells or with bioactive compounds, e.g.,by sequential injection or spraying of substances such as growth factorsor differentiation factors.

A scaffold or scaffold device is the physical structure upon which orinto which cells associate or attach, and a scaffold composition is thematerial from which the structure is made. For example, scaffoldcompositions include biodegradable or permanent materials such as thoselisted below. The mechanical characteristics of the scaffold varyaccording to the application or tissue type for which regeneration issought. It is biodegradable (e.g., collagen, alginates, polysaccharides,polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide) (PLA),or poly(lactide-co-glycolide) (PLGA) or permanent (e.g., silk) In thecase of biodegradable structures, the composition is degraded byphysical or chemical action, e.g., level of hydration, heat or ionexchange or by cellular action, e.g., elaboration of enzyme, peptides,or other compounds by nearby or resident cells. The consistency variesfrom a soft/pliable (e.g., a gel) to glassy, rubbery, brittle, tough,elastic, stiff. The structures contain pores, which are nanoporous,microporous, or macroporous, and the pattern of the pores is optionallyhomogeneous, heterogenous, aligned, repeating, or random.

Alginates are versatile polysaccharide based polymers that may beformulated for specific applications by controlling the molecularweight, rate of degradation and method of scaffold formation. Couplingreactions can be used to covalently attach bioactive epitopes, such asthe cell adhesion sequence RGD to the polymer backbone. Alginatepolymers are formed into a variety of scaffold types. Injectablehydrogels can be formed from low MW alginate solutions upon addition ofa cross-linking agent, such as calcium ions, while macroporous scaffoldsare formed by lyophilization of high MW alginate discs. Differences inscaffold formulation control the kinetics of scaffold degradation.Release rates of morphogens or other bioactive substances from alginatescaffolds are controlled by scaffold formulation to present morphogensin a spatially and temporally controlled manner. This controlled releasenot only eliminates systemic side effects and the need for multipleinjections, but can be used to create a microenvironment that activateshost cells at the implant site.

The scaffold comprises a biocompatible polymer matrix that is optionallybiodegradable in whole or in part. A hydrogel is one example of asuitable polymer matrix material. Examples of materials which can formhydrogels include polylactic acid, polyglycolic acid, PLGA polymers,alginates and alginate derivatives, gelatin, collagen, agarose, naturaland synthetic polysaccharides, polyamino acids such as polypeptidesparticularly poly(lysine), polyesters such as polyhydroxybutyrate andpoly-epsilon-caprolactone, polyanhydrides; polyphosphazines, poly(vinylalcohols), poly(alkylene oxides) particularly poly(ethylene oxides),poly(allylamines)(PAM), poly(acrylates), modified styrene polymers suchas poly(4-aminomethylstyrene), pluronic polyols, polyoxamers,poly(uronic acids), poly(vinylpyrrolidone) and copolymers of the above,including graft copolymers.

The scaffolds are fabricated from a variety of synthetic polymers andnaturally-occurring polymers such as, but not limited to, collagen,fibrin, hyaluronic acid, agarose, and laminin-rich gels. One preferredmaterial for the hydrogel is alginate or modified alginate material.Alginate molecules are comprised of (1-4)-linked β-D-mannuronic acid (Munits) and α L-guluronic acid (G units) monomers, which can vary inproportion and sequential distribution along the polymer chain. Alginatepolysaccharides are polyelectrolyte systems which have a strong affinityfor divalent cations (e.g. Ca⁺², Mg⁺², Ba⁺²) and form stable hydrogelswhen exposed to these molecules. See Martinsen A., et al., Biotech. &Bioeng., 33 (1989) 79-89.) For example, calcium cross-linked alginatehydrogels are useful for dental applications, wound dressingschondrocyte transplantation and as a matrix for other cell types.

An exemplary device utilizes an alginate or other polysaccharide of arelatively low molecular weight, preferably of size which, afterdissolution, is at the renal threshold for clearance by humans, e.g.,the alginate or polysaccharide is reduced to a molecular weight of 1000to 80,000 daltons. Preferably, the molecular mass is 1000 to 60,000daltons, particularly preferably 1000 to 50,000 daltons. It is alsouseful to use an alginate material of high guluronate content since theguluronate units, as opposed to the mannuronate units, provide sites forionic crosslinking through divalent cations to gel the polymer. U.S.Pat. No. 6,642,363, incorporated herein by reference discloses methodsfor making and using polymers containing polysachharides such asalginates or modified alginates that are particularly useful for celltransplantation and tissue engineering applications.

Useful polysaccharides other than alginates include agarose andmicrobial polysaccharides such as those listed in the table below.

Polysaccharide Scaffold Compositions Polymers^(a) Structure FungalPullulan (N) 1,4-;1,6-α-D-Glucan Scleroglucan (N) 1,3;1,6-α-D-GlucanChitin (N) 1,4-β-D-Acetyl Glucosamine Chitosan (C)1,4-β.-D-N-Glucosamine Elsinan (N) 1,4-;1,3-α-D-Glucan Bacterial Xanthangum (A) 1,4-β.-D-Glucan with D-mannose; D-glucuronic Acid as side groupsCurdlan (N) 1,3-β.-D-Glucan (with branching) Dextran (N) 1,6-α-D-Glucanwith some 1,2;1,3-; 1,4-α-linkages Gellan (A) 1,4-β.-D-Glucan withrhamose, D-glucuronic acid Levan (N) 2,6-β-D-Fructan with someβ-2,1-branching Emulsan (A) Lipoheteropolysaccharide Cellulose (N)1,4-β-D-Glucan ^(a)N-neutral, A = anionic and C = cationic.

The scaffolds of the invention are porous or non-porous. For example,the scaffolds are nanoporous having a diameter of less than about 10 nm;microporous wherein the diameter of the pores are preferably in therange of about 100 nm-20 μm; or macroporous wherein the diameter of thepores are greater than about 20 μm, more preferably greater than about100 μm and even more preferably greater than about 400 μm. In oneexample, the scaffold is macroporous with aligned pores of about 400-500μm in diameter. Other methods of preparing porous hydrogel products areknown in the art. (U.S. Pat. No. 6,511,650 incorporated herein byreference).

Bioactive Compositions

The device includes one or more bioactive compositions. Bioactivecompositions are purified naturally-occurring, synthetically produced,or recombinant compounds, e.g., polypeptides, nucleic acids, smallmolecules, or other agents. The compositions described herein arepurified. Purified compounds are at least 60% by weight (dry weight) thecompound of interest. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight thecompound of interest. Purity is measured by any appropriate standardmethod, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

The bioactive composition comprises an element to improve a function ofthe scaffold composition or to promote angiogenesis. For example, atleast one cell adhesion molecule is incorporated into or onto thepolymer matrix to attach the scaffold composition to the local tissuesite and prevent diffusion of the device. Such molecules areincorporated into the polymer matrix prior to polymerization of thematrix or after polymerization of the matrix. Examples of cell adhesionmolecules include, but are not limited to, peptides, proteins andpolysaccharides. More specifically, cell adhesion molecules include, butare not limited to, fibronectin, laminin, collagen, thrombospondin 1,vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein,cartilage matrix protein, fibrinogen, fibrin, fibulin, mucins, entactin,osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin,versican, von Willebrand Factor, polysaccharide heparin sulfate,connexins, collagen, RGD (Arg-Gly-Asp) and YIGSR (Tyr-Ile-Gly-Ser-Arg)(SEQ ID NO: 9) peptides and cyclic peptides, glycosaminoglycans (GAGs),hyaluronic acid (HA), condroitin-6-sulfate, integrin ligands, selectins,cadherins and members of the immunoglobulin superfamily. Other examplesinclude neural cell adhesion molecules (NCAMs), intercellular adhesionmolecules (ICAMs), vascular cell adhesion molecule (VCAM-1),platelet-endothelial cell adhesion molecule (PECAM-1), L1, and CHL1.

Examples of some of these molecules and their function are shown in thefollowing table.

ECM Proteins and peptides and role in cell function Seq. ID ProteinSequence No: Role Fibronectin RGDS Adhesion LDV Adhesion REDV AdhesionVitronectin RGDV Adhesion Laminin A LRGDN 7 Adhesion IKVAV 8Neurite extension Laminin B1 YIGSR 9 Adhesion of many cells, via67 kD laminin receptor PDSGR 10 Adhesion Laminin B2 RNIAEIIKDA 11Neurite extension Collagen 1 RGDT Adhesion of most cells DGEAAdhesion of platelets, other cells Thrombo- RGD Adhesion of most cellsspondin VTXG Adhesion of platelets Hubbell, J A (1995): Biomaterials intissue engineering. Bio/Technology 13: 565-576. One-letter abbreviationsof amino acids are used, X stands for any amino acid.

Additional examples of suitable cell adhesion molecules are shown below.

Amino acid sequences specific for proteoglycanbinding from extracellular matrix proteins SEQ. SEQUENCE ID. NO. PROTEINXBBXBX* 2 Consensus sequence PRRARV 3 Fibronectin YEKPGSPPREVVPRPRPGV 4Fibronectin RPSLAKKQRFRHRNRKGYRSQRGHSRGR 5 Vitronectin RIQNLLKITNLRIKFVK6 Laminin

Particularly preferred cell adhesion molecules are peptides or cyclicpeptides containing the amino acid sequence arginine-glycine-asparticacid (RGD) which is known as a cell attachment ligand and found invarious natural extracellular matrix molecules. A polymer matrix withsuch a modification provides cell adhesion properties to the scaffold,and sustains long-term survival of mammalian cell systems, as well assupporting cell growth.

Coupling of the cell adhesion molecules to the polymer matrix isaccomplished using synthetic methods which are in general known to oneof ordinary skill in the art and are described in the examples.Approaches to coupling of peptides to polymers are discussed in Hiranoand Mooney, Advanced Materials, p. 17-25 (2004). Other useful bondingchemistries include those discussed in Hermanson, BioconjugateTechniques, p. 152-185 (1996), particularly by use of carbodiimidecouplers, DCC and DIC (Woodward's Reagent K). Since many of the celladhesion molecules are peptides, they contain a terminal amine group forsuch bonding. The amide bond formation is preferably catalyzed by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which is a watersoluble enzyme commonly used in peptide synthesis. The density of celladhesion ligands, a critical regulator of cellular phenotype followingadhesion to a biomaterial. (Massia and Hubbell, J. Cell Biol.114:1089-1100, 1991; Mooney et al., J. Cell Phys. 151:497-505, 1992; andHansen et al., Mol. Biol. Cell 5:967-975, 1994) can be readily variedover a 5-order of magnitude density range.

Device Construction

The scaffold structure is constructed out of a number of differentrigid, semi-rigid, flexible, gel, self-assembling, liquid crystalline,or fluid compositions such as peptide polymers, polysaccharides,synthetic polymers, hydrogel materials, ceramics (e.g., calciumphosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans,metals and metal alloys. The compositions are assembled into cellscaffold structures using methods known in the art, e.g., injectionmolding, lyophillization of preformed structures, printing,self-assembly, phase inversion, solvent casting, melt processing, gasfoaming, fiber forming/processing, particulate leaching or a combinationthereof. The assembled devices are then implanted or administered to thebody of an individual to be treated.

The device is assembled in vivo in several ways. The scaffold is madefrom a gelling material, which is introduced into the body in itsungelled form where it gels in situ. Exemplary methods of deliveringdevice components to a site at which assembly occurs include injectionthrough a needle or other extrusion tool, spraying, painting, or methodsof deposit at a tissue site, e.g., delivery using an application deviceinserted through a cannula. In one example, the ungelled or unformedscaffold material is mixed with bioactive substances and cells prior tointroduction into the body or while it is introduced. The resultant invivo/in situ assembled scaffold contains a mixture of these substancesand cells.

In situ assembly of the scaffold occurs as a result of spontaneousassociation of polymers or from synergistically or chemically catalyzedpolymerization. Synergistic or chemical catalysis is initiated by anumber of endogenous factors or conditions at or near the assembly site,e.g., body temperature, ions or pH in the body, or by exogenous factorsor conditions supplied by the operator to the assembly site, e.g.,photons, heat, electrical, sound, or other radiation directed at theungelled material after it has been introduced. The energy is directedat the scaffold material by a radiation beam or through a heat or lightconductor, such as a wire or fiber optic cable or an ultrasonictransducer. Alternatively, a shear-thinning material, such as anampliphile, is used which re-cross links after the shear force exertedupon it, for example by its passage through a needle, has been relieved.

Suitable hydrogels for both in vivo and ex vivo assembly of scaffolddevices are well known in the art and described, e.g., in Lee et al.,2001, Chem. Rev. 7:1869-1879. The peptide amphiphile approach toself-assembly assembly is described, e.g., in Hartgerink et al., 2002,Proc. Natl. Acad. Sci. U.S.A. 99:5133-5138. A method for reversiblegellation following shear thinning is exemplified in Lee et al., 2003,Adv. Mat. 15:1828-1832.

A multiple compartment device is assembled in vivo by applyingsequential layers of similarly or differentially doped gel or otherscaffold material to the target site. For example, the device is formedby sequentially injecting the next, inner layer into the center of thepreviously injected material using a needle, forming concentricspheroids. Non-concentric compartments are formed by injecting materialinto different locations in a previously injected layer. A multi-headedinjection device extrudes compartments in parallel and simultaneously.The layers are made of similar or different scaffolding compositionsdifferentially doped with bioactive substances. Alternatively,compartments self-organize based on their hydro-philic/phobiccharacteristics or on secondary interactions within each compartment.

Compartmentalized Device

A compartmentalized device is designed and fabricated using differentcompositions or concentrations of compositions for each compartment. Forexample, a first bioactive composition is encapsulated within hydrogels,using standard encapsulation techniques (e.g., alginate microbeadformation). This compartment is then coated with a second layer of gel(e.g., double layered alginate microbeads). This second compartment isformed from the same material that contains bioactive compositionelements, the same material in a distinct form (e.g., varying mechanicalproperties or porosity), or a completely different material thatprovides appropriate chemical/physical properties.

Alternatively, the compartments are fabricated individually, and thenadhered to each other (e.g., a “sandwich” with an inner compartmentsurrounded on one or all sides with the second compartment). This latterconstruction approach is accomplished using the intrinsic adhesivenessof each layer for the other, diffusion and interpenetration of polymerchains in each layer, polymerization or cross-linking of the secondlayer to the first, use of an adhesive (e.g., fibrin glue), or physicalentrapment of one compartment in the other. The compartmentsself-assemble and interface appropriately, either in vitro or in vivo,depending on the presence of appropriate precursors (e.g., temperaturesensitive oligopeptides, ionic strength sensitive oligopeptides, blockpolymers, cross-linkers and polymer chains (or combinations thereof),and precursors containing cell adhesion molecules that allowcell-controlled assembly). An individual with ordinary skill in the artof stem cell biology and biomaterials can readily derive a number ofpotentially useful designs for combining or separating components of abioactive composition.

Alternatively, the compartmentalized device is formed using a printingtechnology. Successive layers of a scaffold precursor doped withbioactive substances and/or cells is placed on a substrate then crosslinked, for example by self-assembling chemistries. When the crosslinking is controlled by chemical-, photo- or heat-catalyzedpolymerization, the thickness and pattern of each layer is controlled bya masque, allowing complex three dimensional patterns to be built upwhen un-cross-linked precursor material is washed away after eachcatalyzation. (WT Brinkman et al., Photo-cross-linking of type 1collagen gels in the presence of smooth muscle cells: mechanicalproperties, cell viability, and function. Biomacromolecules, 2003July-August; 4(4): 890-895; W. Ryu et al., The construction ofthree-dimensional micro-fluidic scaffolds of biodegradable polymers bysolvent vapor based bonding of micro-molded layers. Biomaterials, 2007February; 28(6): 1174-1184; Wright, Paul K. (2001). 21st Centurymanufacturing. New Jersey: Prentice-Hall Inc.) Complex,multi-compartment layers are also built up using an inkjet device which“paints” different doped-scaffold precursors on different areas of thesubstrate. Julie Phillippi (Carnegie Mellon University) presentation atthe annual meeting of the American Society for Cell Biology on Dec. 10,2006; Print me a heart and a set of arteries, Aldhouse P., New Scientist13 Apr. 2006 Issue 2547 p 19; Replacement organs, hot off the press, C.Choi, New Scientist, 25 Jan. 2003, v2379. These layers are built-up intocomplex, three dimensional compartments. The device is also built usingany of the following methods: Jetted Photopolymer, Selective LaserSintering, Laminated Object Manufacturing, Fused Deposition Modeling,Single Jet Inkjet, Three Dimensional Printing, or Laminated ObjectManufacturing.

Growth Factors and Incorporation of Compositions into/onto a ScaffoldDevice

Bioactive substances that influence growth, development, movement, andother cellular functions are introduced into or onto the scaffoldstructures. Such substances include BMP, bone morphogenetic protein;ECM, extracellular matrix proteins or fragments thereof; EGF, epidermalgrowth factor; FGF-2, fibroblast growth factor 2; NGF, nerve growthfactor; PDGF, platelet-derived growth factor; PIGF, placental growthfactor; TGF, transforming growth factor, and VEGF, vascular endothelialgrowth factor. Cell-cell adhesion molecules (cadherins, integrins,ALCAM, NCAM, proteases) are optionally added to the scaffoldcomposition.

Exemplary growth factors and ligands are provided in the tables below.

Growth factors used for angiogenesis Growth factor Abbreviation Relevantactivities Vascular VEGF Migration, proliferation and endothelialsurvival of ECs growth factor Basic fibroblast bFGF-2 Migration,proliferation and survival of growth factor ECs and many other celltypes Platelet-derived PDGF Promotes the maturation of blood growthfactor vessels by the recruitment of smooth muscle cells Angiopoietin-1Ang-1 Strengthens EC-smooth muscle cell interaction Angiopoietin-2 Ang-2Weakens EC-smooth muscle cell interaction Placental PIGF Stimulatesangiogenesis growth factor Transforming TGF Stabilizes new blood vesselsby growth factor promoting matrix deposition

Growth factors used for wound healing Growth Factor AbbreviationRelevant activities Platelet-derived PDGF Active in all stages ofhealing process growth factor Epidermal growth EGF Mitogenic forkeratinocytes factor Transforming TGF-β Promotes keratinocyte migration,growth factor-β ECM synthesis and remodeling, and differentiation ofepithelial cells Fibroblast growth FGF General stimulant for woundhealing factor

Growth Factors Used for Tissue-Engineering Moleular Representativeweight supplier of rH growth Growth factor Abbreviation (kDa) Relevantactivities factor Epidermal growth factor EGF 6.2 Proliferation ofepithelial, mesenchymal, PeproTech Inc. and fibroblast cells (RockyHill, NJ, USA) Platelet-derived PDGF-AA 28.5 Proliferation andchemoattractant agent for PeproTech Inc. growth factor PDGF-AB 25.5smooth muscle cells; extracellular matrix PDGF-BB 24.3 synthesis anddeposition Transforming TFG-α 5.5 Migration and proliferation ofPeproTech Inc. growth factor-α keratinocytes; extracellular matrixsynthesis and deposition Transforming TGF-β 25.0 Proliferation anddifferentiation of bone PeproTech Inc. growth factor-β forming cells;chemoattractant for fibroblasts Bone BMP-2 26.0 Differentiation andmigration of bone Cell Sciences Inc. morphogenetic BMP-7 31.5 formingcells (Norwood, MA, USA) protein Basic fibroblast bFGF/FGF-2 17.2Proliferation of fibroblasts and initiation of PeproTech Inc. growthfactor angiogenesis Vascular endothelial VEGF₁₆₅ 38.2 Migration,proliferation, and survival of PeproTech Inc. growth factor endothelialcells rH, recombinant human

Immobilized ligands used in tissue engineering Immobilized ligand* ECMmolecule source Application RGD Multiple ECM molecules, Enhance bone andcartilage tissue formation in including fibronectin, vitro and in vivovitronectin, laminin, collagen Regulate neurite outgrowth in vitro andin vivo and thrombospondin Promote myoblast adhesion, proliferation anddifferentiation Enhance endothelial cell adhesion and proliferationIKVAV (SEQ ID NO: 8), YIGSR Laminin Regulate neurite outgrowth in vitroand in vivo (SEQ ID NO: 9), RNIAEIIKDI (SEQ ID NO: 11) Recombinantfibronectin fragment Fibronectin Promote formulation of focal contactsin pre- (FNIII₇₋₁₀) osteoblasts Ac-GCRDGPQ-GIWGQDRCG Common MMPsubstrates, Encourage cell-mediated proteolytic degradation, (SEQ ID NO:18) (e.g. collagen, fibronectin, remodeling and bone regeneration (withRGD and laminin) BMP-2 presentation) in vivo *Sequences are given insingle-letter amino acid code. MMP, matrix metalloproteinase.

The release profiles of bioactive substances from scaffold devices iscontrolled by both factor diffusion and polymer degradation, the dose ofthe factor loaded in the system, and the composition of the polymer.Similarly, the range of action (tissue distribution) and duration ofaction, or spatiotemporal gradients of the released factors areregulated by these variables. The diffusion and degradation of thefactors in the tissue of interest is optionally regulated by chemicallymodifying the factors (e.g., PEGylating growth factors). In both cases,the time frame of release determines the time over which effective celldelivery by the device is desired.

Carrier systems for tissue regeneration are described in the tablebelow.

Polymeric carriers used to deliver various growth factors and the typeof tissues regenerated Growth factor Carrier Tissue regenerated EGFGelatin Dermis PET suture Tendon PVA sponge Dermis PDGF Chitosan-PLLAscaffold Craniofacial bone CMC gel Dermis Fibrin Ligament Porous HA LongBone TGF-β Alginate Cartilage PLA Long Bone CaP-titanium meshCraniofacial bone Polyoxamer; PEO gel Dermis rhBMP-2 Collagen spongeLong bone Craniofacial bone HA-TCP granules Spinal bone HA-collagen Longbone PLA-DX-PEG Ectopic and hip bone rHBMP-7 HA Spinal bone Collagen-CMCSpinal bone Porous HA Craniofacial bone bFGF Chitosan DermisHeparin-alginate Blood vessels EVAc microspheres Blood vessels Fibrinmatrices Blood vessels VEGF PLG scaffold Blood vessels PLG scaffoldBlood vessels PLG microspheres Blood vessels Fibrin mesh Blood vesselsAbbreviations: PET, poly (ethylene terepthalate); PVA, polyvinylalcohol; PLLA, poly(L-lactic acid); CMC, carboxymethylcellulose; HA,hydroxyapatite; PLA, poly(D,L-lactic acid); CaP, calcium phosphate; PEO,poly (ethylene oxide); TCP, tricalcium phosphate; PEG, poly(ethyleneglycol); -DX-, -p-dioxanone-; EVAc, ethylene vinyl acetate; PLG, poly(lactide-co-glycolide).

The bioactive substances are added to the scaffold compositions usingknown methods including surface absorption, physical immobilization,e.g., using a phase change to entrap the substance in the scaffoldmaterial. For example, a growth factor is mixed with the scaffoldcomposition while it is in an aqueous or liquid phase, and after achange in environmental conditions (e.g., pH, temperature, ionconcentration), the liquid gels or solidifies thereby entrapping thebioactive substance. Alternatively, covalent coupling, e.g., usingalkylating or acylating agents, is used to provide a stable, long-termpresentation of a bioactive substance on the scaffold in a definedconformation. Exemplary reagents for covalent coupling of suchsubstances are provided in the table below.

Methods to covalently couple peptides/proteins to polymers FunctionalReacting groups Group of on proteins/ Polymer Coupling reagents andcross-linker peptides —OH Cyanogen bromide (CNBr) —NH₂ Cyanuric chloride4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4- methyl-morpholinium chloride(DMT-MM) —NH₂ Diisocyanate compounds —NH₂ Diisothoncyanate compounds —OHGlutaraldehyde Succinic anhydride —NH₂ Nitrous Acid —NH₂ Hydrazine +nitrous acid —SH —Ph—OH —NH₂ Carbodiimide compounds (e.g., EDC, —COOHDCC)[a] DMT-MM —COOH Thionyl chloride —NH₂ N-hydroxysuccinimideN-hydroxysulfosuccinimide + EDC —SH Disulfide compound —SH [a]EDC:1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; DCC:dicyclohexylcarbodiimide

The following materials and methods were used to generate the datadescribed herein.

Preparation and Loading of Polymer/Gel Compositions

Ultrapure alginates were purchased from ProNova Biomedical (Norway). MVGalginate, a high-G-containing alginate (M/G ratio of 40/60 as specifiedby the manufacturer), was used as the high molecular weight (molecularmass=250,000 Da) component to prepare gels. Low molecular weightalginate (molecular mass=50,000 Da) was obtained by gamma-irradiatinghigh molecular weight alginate with a cobalt-60 source for 4 h at agamma-dose of 3.0 Mrad (Radiation Lab, Massachusetts Institute ofTechnology). The alginate used to form gels was a combination of the twodifferent molecular weight polymers at a ratio of 3:1. Both alginatepolymers were diluted to 1% w/v in double-distilled H₂0, and 1% of thesugar residues in the polymer chains were oxidized with sodium periodate(Aldrich, St Louis, Mo.) by maintaining solutions in the dark for 17 hat room temperature. An equimolar amount of ethylene glycol (Fisher,Pittsburgh, Pa.) was added to stop the reaction, and the solution wassubsequently dialyzed (MWCO 1000, Spectra/Por, Rancho Dominguez, Calif.)over 3 days. The solution was sterile filtered, frozen (−20 degree C.overnight), lyophilized and stored at −20 degree C. To prepare gels,modified alginates were reconstituted in EBM-2 (Lonza, Walkersville,Md.) to obtain a 2% w/v solution(75% low molecular weight, 25% highmolecular weight MVG used in all experiments) prior to gelation. The 2%w/v alginate solutions were cross-linked with aqueous slurries of acalcium sulfate solution (0.21 g CaS0₄ mL/L distilled H20) at a ratio of25:1 (40 microliter of CaS04 per 1 mL of 2% w/v alginate solution) usinga 1 mL syringe. Reconstituted alginate was stored at 4 degree C. Forincorporation of VEGF, PDGF and DAPT, alginate solutions were mixed withrecombinant human VEGF 165, PDGF-BB (R&D systems, MN) or DAPT(N—[N-(3,5-Difluorophenacetyl-L-alanyl)]-Sphenylglycine t-Butyl Ester)(EMD Chemicals, NJ) by using two syringes coupled by a syringeconnector. Calcium slurry (Sigma, St Louis, Mo.) was then mixed with theresulting alginate solution using two syringes coupled by a syringeconnector to facilitate the mixing process and prevent entrapment of airbubbles during mixing. The mixture was allowed to gel for 30 min, andthen was maintained at 4 degrees C. prior to animal injections.

Murine Hindlimb Model of Ischemia

The animals used were 6-week old severe combined immunodeficiency (SCID)mice on a C57BL/6J background (Jackson Laboratory, ME). Unilateralhindlimb ischemia was created as follows. The animals were anesthetizedby intraperitoneal injections of ketamine (80 mg/kg) and xylazine (5mg/kg). The external iliac and femoral artery and vein were ligated, and50 μL alginate hydrogel incorporating 3 μg VEGF and/or 86-8600 ng DAPTwas injected near the distal end of the ligation site. As controls, VEGFand DAPT in PBS were also injected intramuscularly or intraperitoneally(bolus delivery). Incisions were closed by 5-0 Ethicon sutures (Johnson& Johnson, N.J.). Blood flow in the hindlimb was monitored by a laserDoppler perfusion imaging (LDP/) system (Perimed AB, Sweden), and theresults were normalized to the control unligated limb of the sameanimal.

Histology and Immunohistochemistry

Hindlimb muscle tissues between the two suture knots defining theligation site were dissected and fixed by Z-fix (Anatech, M/) overnightand changed into 70% EtOH for storage prior to histology processing.Samples were embedded in paraffin and sectioned (5 μm thick) onto slidesby Paragon (Paragon Bioservices, MD). Sections were incubated withprimary anti-mouse CD31 antibody (1:250) (Pharmingen, Calif.) orα-smooth muscle actin antibody, followed by incubation with an anti-ratmouse biotinylated secondary (1:200) (Vector Laboratories, CA), andamplified by a Tyramide Signal Amplification (TSA) Biotin System (PerkinElmer Life Sciences, MA). Staining was developed using DAB+ substratechromogen (DAKO, CA) and counterstained with Mayer's Hematoxylin.Capillary densities were quantified by counting the CD31 positivecapillary numbers, normalized to the tissue area, in 30 randomly chosenhigh-power (200×, 400×) fields. Images were captured with anOlympus-IX81 light microscope connected to an Olympus DP70 digital imagecapture system.

EXAMPLE 1 In vitro Model to Test the Significance of a Controlled LocalConcentration of VEGF

Endothelial cells isolated from diabetics are shown to have a reducedresponse to VEGF as compared to age-matched non-diabetics (FIG. 1,*P<0.05), as reflected in their capability of forming sprouts, the firststep in angiogenesis. In addition, there is an optimal VEGFconcentration to induce most sprouts. This suggests the need of acontrolled concentration of growth factors in the local area to reachthe best therapeutic effect.

EXAMPLE 2 In vitro Model to Test the Significance of a Combination ofDAPT and VEGF

Endothelial cells isolated from diabetics produce more sprouts in thepresence of a combination of both gamma-secretase inhibitors with VEGF.(FIG. 2, *P<0.05) than with either of them alone, which implies the needof a combination of both growth factor and DAPT.

EXAMPLE 3 In vitro Model Establishing the Significance of a DistinctPresentation of VEGF and DAPT

This examples illustrates the concept that while the combination of VEGFand DAPT is superior to either single factor alone, the optimalconcentration for each individual compound does not coincide. As shownin FIG. 3 (*P<0.05), the concentration of VEGF that gives the mostsprouts if used alone (50 ng/ml) is less superior to a lowerconcentration (10 ng/ml) when in combined use with DAPT (2.5 μM).Moreover, increasing both the concentration of VEGF (50 ng/ml) and DAPT(10 μM) actually reduces the number of sprouts. This indicates that thepresentation of VEGF and DAPT may need to be separately controlled toachieve an optimal effect.

EXAMPLE 4 In vivo Model to Test the Effect of Controlled Presentation ofVEGF and DAPT to Recover Blood Flow in an Ischemia Situation

Alginate is used as the delivery vehicle. Injectable alginate hydrogelsincorporating VEGF and GSI is injected into the hindlimb ischemia sitecreated by femoral artery and vein ligation in a murine model. Releaseprofiles of VEGF and DAPT from alginate hydrogels are distinct, as shownin FIG. 4A. Blood flow before and after ligation surgery is measured bylaser doppler perfusion imaging (LDPI) to indicate the extent ofangiogenesis in the ischemia area. As shown in FIGS. 4B and 4C,(*P<0.05), when DAPT and VEGF are both incorporated in the alginatehydrogel the blood flow recovery is superior to simple bolus injectionof them together, either drug alone from alginate gel or by bolusinjection (including direct intramuscular and intraperitonealinjection), and blank control. This points to the significance of acontrolled presentation of multiple pro-angiogenic compounds as comparedto bolus injection.

EXAMPLE 5 In vivo Model to Test the Effect of Controlled Presentation ofVEGF and DAPT on Newly-Formed Blood Vessel Density in an IschemiaSituation

A combination of VEGF and DAPT delivered by alginate gel systems gaverise to the highest blood vessel density as compared to blank control,either alone released from gels (FIG. 5A), or administered viaintramuscular or intraperitoneal injection (FIG. 5B). These dataindicate the significance of controlled delivery of both VEGF and DAPTby the alginate gel system, i.e. controlled and coordinated release ofgrowth factors and signaling molecules leads to an improved clinicalresult compared to conventional delivery methods.

EXAMPLE 6 In vivo Evaluation of the Effect of DAPT Delivered FromAlginate Gel System and From Intraperitoneal Injection on the Cells inSmall Intestines

As compared to normal controls, DAPT delivered from alginate gel systemdoes not alter cell differentiation at distant sites as much as DAPTdelivered via intraperitoneal injection (DAPT delivered via injectiondisrupts normal intestinal structure, as indicated by H&E staining,presence of extra glycosaminoglycans by alcian blue staining, loss ofproliferating cells by Ki67 staining, and downregulation of Notch targetgene expression by HES-1 staining) DAPT delivered from the gel systemonly has effects in the local region while DAPT delivered viaintraperitoneal injection goes into the systemic circulation and led toadverse effects at distant organs (e.g., small intestines as anexample). These data indicate that a controlled local but not systemicpresentation of delivered DAPT is a preferred delivery method.

EXAMPLE 7 Blood Vessel Density and Blood Flow Recovery

In vivo evaluation of the effect of controlled presentation of VEGF andDAPT on newly formed blood vessel density and blood flow recovery wascarried out in an ischemic type I diabetic mouse model. The results(FIGS. 7A-B) indicate that a combination of controlled delivery of aprescribed previously determined ratio, e.g., an optimal level, of VEGFand DAPT increased the blood vessel density and recovered blood flow.

EXAMPLE 8 Comparison of Controlled Presentation of Factors Compared toDelivery of Factors Alone

The effect of controlled presentation of VEGF and PDGF on newly formedblood vessel density and blood flow recovery was evaluated in vivo usingthe ischemic type I diabetic mouse model. The results (FIGS. 8A-B)indicate that a combination of optimal level of VEGF and PDGF issuperior to VEGF or PDGF alone in recovering blood flow.

EXAMPLE 9 Controlled Presentation of a Combination of Factors VEGF, PDGFand DAPT on Angiogenesis

In vivo evaluation of the effect of controlled presentation of VEGF,PDGF and DAPT on newly formed blood vessel density and blood flowrecovery was carried out in an ischemic type I diabetic mouse model.These results (FIGS. 9A-B) indicate that a combination of optimal levelof VEGF and DAPT is superior to a combination of VEGF and PDGF inrecovering blood flow.

EXAMPLE 10 Maturation of Newly Formed Blood Vessels

The effect of controlled presentation of VEGF, PDGF and DAPT on thematuration of newly formed blood vessels was evaluated using the same invivo ischemic type I diabetic mouse model. The result (FIGS. 10A-C)indicates that a combination of optimal level of VEGF PDGF and DAPT issuperior to a combination of VEGF and PDGF in generating more maturedblood vessels.

The data generated described herein indicate that compositions andmethods not only reliably induce and promote angiogenesis in bodilytissues and organs but also promote and support maturation of thosevessels into functional vasculature to improve blood flow to ischemic,damaged, injured, or otherwise compromised tissues and organs.

Other Embodiments

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of inducing growth of new blood vesselseither from existing blood vessels, or creation of de novo vessels, or acombination thereof, in a target tissue of a mammal, comprisingcontacting a mammalian cell by injecting into a tissue a devicecomprising an alginate hydrogel scaffold composition comprisingN-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester(DAPT) and vascular endothelial growth factor (VEGF), wherein saidscaffold composition temporally controls release of the DAPT and theVEGF, wherein the VEGF said bioactive composition induces angiogenesis,arteriogenesis, or vasculogenesis, and wherein the VEGF comprisesVEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉, or VEGF₂₀₆.
 2. The method of claim1, wherein the DAPT is released before the VEGF.
 3. The method of claim1, wherein said VEGF is released over a period of weeks and said DAPT isreleased over a period of less than one week.
 4. The method of claim 1,wherein said mammal is a human.
 5. The method of claim 1, wherein saidmammal has diabetes.
 6. The method of claim 1, wherein said hydrogelscaffold comprises nanopores.
 7. The method of claim 1, wherein the DAPTis released from the scaffold composition at a first rate and the VEGFis released from the scaffold composition at a second rate, and whereinthe second rate is slower than the first rate.
 8. The method of claim 7,wherein the VEGF exits from the scaffold composition for a time periodof one or more weeks.
 9. The method of claim 7, wherein the DAPT exitsfrom the scaffold composition for a period of one or more days.
 10. Themethod of claim 1, wherein the device comprises a molar ratio of 1:1 to1:200 for VEGF₁₆₅ to DAPT.
 11. The method of claim 10, wherein thedevice comprises a molar ratio of 1:31 for VEGF₁₆₅ to DAPT.
 12. Themethod of claim 1, wherein the mammal has coronary arterial disease(CAD) or peripheral arterial disease (PAD).
 13. The method of claim 1,wherein the mammal has acute or chronic ischemia due to peripheralarterial disease (PAD).
 14. The method of claim 1, wherein the mammalhas a diabetic ulcer or a wound due to peripheral arterial disease(PAD).
 15. A method of inducing growth of new blood vessels either fromexisting blood vessels, or creation of de novo vessels, or a combinationthereof, in a target tissue of a mammal, comprising contacting amammalian cell by injecting into a tissue a device comprising analginate hydrogel scaffold composition comprisingN-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester(DAPT) and a bioactive composition selected from the group consisting ofvascular endothelial growth factor (VEGF) and platelet-derived growthfactor (PDGF), wherein said scaffold composition temporally controlsrelease of the DAPT and the bioactive composition, wherein the bioactivecomposition induces angiogenesis, arteriogenesis, or vasculogenesis, andwherein the VEGF comprises VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉, orVEGF₂₀₆.
 16. The method of claim 15, wherein said DAPT is releasedwithin 1-3 days and said VEGF or PDGF is released within 7-60 days. 17.The method of claim 15, wherein the DAPT is released before thebioactive composition.
 18. The method of claim 15, wherein the PDGFcomprises PDGF-BB.
 19. The method of claim 15, wherein the devicecomprises a molar ratio of 1:1 to 1:200 for VEGF₁₆₅ to DAPT, or 1:1 to1:200 for PDGF-BB to DAPT.
 20. The method of claim 19, wherein thedevice comprises a molar ratio of 1.8:31 for PDGF-BB to DAPT.
 21. Themethod of claim 15, wherein the mammal has coronary arterial disease(CAD) or peripheral arterial disease (PAD).
 22. The method of claim 15,wherein the mammal has acute or chronic ischemia due to peripheralarterial disease (PAD).
 23. The method of claim 15, wherein the mammalhas a diabetic ulcer or a wound due to peripheral arterial disease(PAD).
 24. A method of inducing growth of new blood vessels either fromexisting blood vessels, or creation of de novo vessels, or a combinationthereof, in a target tissue of a mammal, comprising contacting amammalian cell by injecting into a tissue a device comprising a hydrogelscaffold composition comprisingN-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester(DAPT), vascular endothelial growth factor (VEGF), and platelet-derivedgrowth factor (PDGF), wherein said scaffold composition temporallycontrols release of the DAPT, VEGF, and PDGF, and wherein the VEGF andPDGF induce angiogenesis, arteriogenesis, or vasculogenesis.
 25. Themethod of claim 24, wherein the device comprises a molar ratio of1:0.1:1 to 1:10:200 for VEGF₁₆₅, PDGF-BB and DAPT.
 26. The method ofclaim 25, wherein the device comprises a molar ratio of 1:1.8:31 forVEGF₁₆₅, PDGF-BB and DAPT.
 27. The method of claim 24, wherein themammal has coronary arterial disease (CAD) or peripheral arterialdisease (PAD).
 28. The method of claim 24, wherein the mammal has acuteor chronic ischemia due to peripheral arterial disease (PAD).
 29. Themethod of claim 24, wherein the mammal has a diabetic ulcer or a wounddue to peripheral arterial disease (PAD).